Studying Disease-Associated UBE3A Missense Variants Using Enhanced Sampling Molecular Simulations

Abstract

Missense variants in UBE3A underlie neurodevelopmental conditions such as Angelman Syndrome and Autism Spectrum Disorder, but the underlying molecular pathological consequences on protein folding and function are poorly understood. Here, we report a novel, maternally inherited, likely pathogenic missense variant in UBE3A (NM_000462.4(UBE3A_v001):(c.1841T>C) (p.(Leu614Pro))) in a child that presented with myoclonic epilepsy from 14 months, subsequent developmental regression from 16 months, and additional features consistent with Angelman Syndrome. To understand the impact of p.(Leu614Pro) on UBE3A, we used adiabatic biased molecular dynamics and metadynamics simulations to investigate conformational differences from wildtype proteins. Our results suggest that Leu614Pro substitution leads to less efficient binding and substrate processing compared to wildtype. Our results support the use of enhanced sampling molecular simulations to investigate the impact of missense UBE3A variants on protein function that underlies neurodevelopment and human disorders.

Figure 1

Family pedigree for the affected female subject (arrowhead).

Figure 2

Preparation of UBE3A closed-state structure. Multiple copies of the UBE3A:E2 complex (PDB: 1C4Z) were aligned to relevant portions of the NEDD4-2:E2:ubiquitin complex (PDB: 3JW0), in accordance with each colored segment. The closed-form complex was constructed by generating a chimeric model based on the aligned segments. The loop structure between the N- and C-lobes (residues 737–741) was generated in a template-free manner during model building to ensure an appropriate positioning of this loop relative to prepositioned domains. Legend: transparent gray—NEDD4-2 and E2 from PDB: 3JW0; solid gray—ubiquitin from PDB 3JW0; pink—C-lobe-contacting subdomain of N-lobe of UBE3A (residues 497–626 and 703–756) from PDB: 1C4Z; blue-violet—E2-contacting subdomain of N-lobe of UBE3A (residues 627–702) and E2 from PDB: 1C4Z; green—C-lobe of UBE3A from PDB: 1C4Z (residues 742–846); and yellow—location of Leu614 within the C-lobe-contacting subdomain of the UBE3A N-lobe.

Figure 3

Frames representing the path between open and closed states of UBE3A generated via ABMD. Interframe root-mean-squared deviations are shown above each arrow. Position along path (s) noted underneath each frame. E2 is colored gray, while UBE3A is colored from N-to-C terminal in a blue-to-red rainbow in each frame.

Figure 4

Well-tempered metadynamics simulations of wildtype and Leu614Pro UBE3A-E2 complexes. (A) Free energy surface for the wildtype UBE3A-E2 complex. (B–E) Structural ensembles associated with low-energy states of the wildtype UBE3A-E2 complex. (F) Free energy surface for the UBE3A-E2 complex. (G–J) Structural ensembles associated with low-energy states of the UBE3A-E2 complex. The approximate locations of the structural ensembles in the free energy surfaces are marked with the corresponding panel identifier and the structures have been aligned with respect to E2 and shown with the same camera position in all depictions. Legend for structural ensembles: gray—E2; blue-to-red rainbow—UBE3A N-to-C terminal.